Optical microscopes in which a point or, more exactly, a very small volume (say 1 μm3 or less, e.g. of the order of 0.01 μm3) of the specimen is illuminated at any one time have been developed for a number of different optical imaging methods, including confocal epifluorescence imaging (White, U.S. Pat. No. 5,032,720) and multiphoton imaging (Denk, Strickler and Webb, U.S. Pat. No. 5,034,613). Some of these methods are reviewed in the Handbook of Biological Confocal Microscopy, edited by James B. Pawley, Second Edition, Plenum Press (1995). In the case of a so-called scanning spot microscope, an image is built up by scanning the illumination spot over the specimen or by scanning the specimen relative to a stationary spot, some portion of the emitted or reflected radiation being measured over a period of time and used to determine the intensity of the image at each point. In an alternative form, a broad area of the specimen is illuminated and it is the photometric area or volume which is scanned. The latter scheme is particulary applicable to microdensitometry at single or multiple optical wavelengths. As described in the Handbook of Biological Confocal Microscopy, such instruments have found widespread application, particularly in biological studies using confocal or multiphoton epifluorescence microscopy where the intensity of the measured light is often very low. In such a situation, loss of light in filters or monochromators, which are frequently used to isolate light of specific wavelengths before passage to the detector, is a serious problem.
In the field of astronomical spectroscopy a method for analyzing the spectral composition of the light is described by L. Mertz (Spectrometre Stellaire Multicanal. Le Journal de Physique et le Radium Vol. 19, 233-235, 1958). Mertz refers to two advantages of his apparatus. First, insofar as it is a Fourier interferometric device, it is superior to a monochromator. This is a reference to the well-known fact that in a monochromator light is wasted on the jaws of the exit slit, whereas in an interferometer all photons of light are potentially detectable. Secondly, he states that his apparatus, which depends on the use of birefringent components, is superior in robustness and simplicity to the alternative of a Michelson interferometer.
At first sight, an objection to Mertz's apparatus is that since it requires a polarized input, at least half of the intensity of the incident light is sacrificed. However, Mertz introduces a principle which might be termed “full-polarization usage”. The input starlight is passed through a Wollaston prism which acts as a polarizing polarising beamsplitter, dividing the input into two beams with mutually perpendicular polarizations with minimum wastage of light. The two beams pass through the polarizing interferometer in such a way that two complementary interferograms are obtained, both of which are used for subsequent analysis. This feature has been further developed by W. M. Sinton who incorporates two Wollaston prisms respectively as polarizing beamsplitter and combiner (W. M. Sinton, Recent Infrared Spectra of Mars and Venus, Journal of Quantitative Spectroscopy and Radiation Transfer, Vol 3, 551-558, 1963). The methods of Mertz and Sinton both depend on making series of measurements of the intensity produced by the polarizing interferometer at different settings of optical path difference between two beams (the two being produced as a result of the birefringence in the optical materials). Such a series of measurements, called an interferogram, is then subjected to an inverse Fourier transformation according to well-established practice, to yield the optical spectrum of the incident light.
Prior art also exists describing the application to a microscope of birefringent apparatus for Fourier spectral analysis. Minami (S. Minami, Fourier Transform Spectroscopy using Image Sensors, Mikrochimika Acta [Wien], III, 309-324, 1987) describes an apparatus in which light from a restricted region of a microscope specimen passes through a slit-shaped aperture and is then allowed to pass through birefringent components such that a complete interferogram is instantaneously produced in a camera.
Buican and Martin (U.S. Pat. No. 4,905,169) disclose a polarizing interferometer for use with a flow cytometer. Their interferometer incorporates the use of two polarizing beamsplitters to achieve maximum use of signal photons, as per Mertz and Sinton, but employs a modulatable birefringent device. This last feature allows a complete interferometric scan (i.e. over a range of optical path differences) to be performed in 0.01 to 0.1 millisecond such that a full spectrum can be obtained within the very short passage time of a single cell through the laser beam in the flow chamber.
The prior art works well, but none of it is directed towards the application of interferometry and Fourier spectral analysis to scanning spot microscopy. Mertz and Sinton restrict themselves to astronomical spectroscopy, where the particular problems of scanning spot microscopy are not encountered. Minami does not envisage any sort of scanning arrangement, and his apparatus is not suitable for use with a photomultiplier, which is the preferred form of detector for scanning spot microscopy because of its good response to weak, rapidly-varying signals. Buican is not concerned with imaging and consequently his apparatus is not adapted to scanning spot microscopy. In a later patent (U.S. Pat. No. 5,117,466) Buican and Yoshida describe the application of the same type of interferometer to a laser scanning epifluorescence microscope, preferably a confocal microscope, as part of a system also including a flow cytometer. The method described by Buican and Yoshida for scanning-spot microscopy requires a rapidly-oscillating crystalline element in order to produce a complete interferogram during the pixel dwell time (i.e. the time taken for a region of the specimen corresponding to one picture element in the final image to be scanned). In the present application the method is different: each interferogram is collected by repeated scanning over the same spot at different settings of optical path difference. This slower procedure permits the use of different apparatus with several advantages, in addition to lower cost.